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Of Thrs Expression Following Threonine Starvation In Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6992-6997, July 1996 Biochemistry Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis (mRNA processing/threonyl tRNA synthetase/mRNA stability/antitermination) CIARA&N CONDON, HARALD PUTZER*, AND MARIANNE GRUNBERG-MANAGO Unite Propre de Recherche 9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France Contributed by Marianne Grunberg-Manago, April 1, 1996 ABSTRACT The threonyl-tRNA synthetase gene, thrS, is Changing the specifier codon in the tyrosyl-tRNA synthetase a member of a family of Gram-positive genes that are induced gene, tyrS, from UAC to UUC, switches the specificity of following starvation for the corresponding amino acid by a regulation from tyrosine to phenylalanine (4). Similarly, transcriptional antitermination mechanism involving the cog- changing the UUC codon of the phenylalanyl-tRNA syn- nate uncharged tRNA. Here we show that an additional level thetase gene to ACC permits induction ofpheS by starvation of complexity exists in the control of the thrS gene with the for threonine (8). A second interaction occurs between the mapping of an mRNA processing site just upstream of the NCCA-3' end of the acceptor arm of the uncharged tRNA and transcription terminator in the thrS leader region. The pro- a complementary sequence (UGGN'), which is part of the cessed RNA is significantly more stable than the full-length T-box and is bulged out of the antiterminator structure (4, 5, transcript. Under nonstarvation conditions, or following star- 8). Although the formation of these two interactions is likely vation for an amino acid other than threonine, the full-length to be the critical element in the stabilization of the antitermi- thrS mRNA is more abundant than the processed transcript. nation conformation of the RNA, it is likely that other However, following starvation for threonine, the thrS mRNA tRNA-leader interactions and possibly some protein factors exists primarily in its cleaved form. This can partly be are required to make this a productive complex in vivo (4, 8). attributed to an increased processing efficiency following We have been studying the mechanism of tRNA-mediated threonine starvation, and partly to a further, nonspecific antitermination in the control of the thrS gene, one of two increase in the stability of the processed transcript under threonyl-tRNA synthetase genes in Bacillus subtilis. Here we starvation conditions. The increased stability ofthe processed show that the thrS leader mRNA is cleaved between the T-box RNA contributes significantly to the levels of functional RNA and the leader terminator structure, and that the cleaved observed under threonine starvation conditions, previously transcript is significantly more stable than the full-length RNA, attributed solely to antitermination. Finally, we show that particulary under starvation conditions. Thus, termination/ processing is likely to occur upstream of the terminator in the antitermination is not the only determinant of the level of leader regions of at least four other genes of this family, functional thrS mRNA in the cell. We show that while the suggesting a widespread conservation of this phenomenon in interaction between uncharged tRNA-Thr and the thrS leader their control. is not necessary for processing to occur, the cleavage efficiency is nonetheless stimulated by threonine starvation. Finally, we Unlike in Escherichia coli, where the genes encoding the show that cleavage between the T-box and the terminator aminoacyl-tRNA synthetases and the amino acid biosynthetic probably occurs in several other members of this family of operons are induced by a wide variety of mechanisms in genes, suggesting that this phenomenon may have a general response to starvation for their cognate amino acid (1, 2), importance in their regulation. many of these genes appear to be regulated by a common transcription antitermination mechanism in Bacillus subtilis and other Gram-positive bacteria (3-6). The genes of this MATERIALS AND METHODS family all have a highly structured leader region of about 300 Bacterial Strains and Culture Conditions. The B. subtilis nucleotides (nt), followed by a Rho-independent transcription strain used for these studies was 168, or derivatives thereof, terminator, just upstream of the translation initiation site. containing thrS-lacZ transcriptional fusions integrated into Upstream of the leader transcription terminator is a highly the amy locus. Strains were in general grown in M9 minimal conserved sequence of about 14 nt, known as the T-box, and medium (9) at 37°C to an OD600 of about 1.0 before harvesting part of the T-box sequence can base pair with a complemen- for RNA isolation. For starvation experiments, cells were tary sequence in the 5' strand of the terminator stem to form grown to an OD600 of about 0.3 in M9 medium, divided, and an alternative, but much weaker, antiterminator structure (2, then starved for 2 h in the presence of either 600 ,ug of 4). The antitermination conformation of the mRNA is thought threonine hydroxamate per ml (Sigma) or 3 ,ug of arginine to be stabilized in each case by the cognate uncharged tRNA, hydroxamate per ml (Sigma), each of which caused about a thus accounting for the induction of these genes by starvation two-fold decrease in growth rate. for their corresponding amino acid (4, 7, 8). Plasmid Construction. The wild-type thrS-lacZ fusion plas- The identity of the stabilizing tRNA is specified by a codon mid (pHMS69) was constructed by cloning a 0.44-kb EcoRI- bulged out of a conserved secondary structure called the BamHI fragment from pHMS25 (8) into the lacZ fusion specifier domain, which occurs early in the leader regions of vector, pHM2 (10), and cut with EcoRI and BamHI. these genes (4, 8). The tRNA is thought to recognize the RNA Isolation. Frozen pellets ofcells (from 20 ml of culture) bulged triplet via a classical codon/anticodon interaction. were resuspended in 4 ml ice-cold TE buffer (pH 8.0). RNA The publication costs of this article were defrayed in part by page charge Abbreviation: RT, reverse transcriptase. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 6992 Downloaded by guest on September 26, 2021 Biochemistry: Condon et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6993 was isolated by vortex mixing the cells in an ice-cold mixture nuclease mapping experiments on the same RNA preparation. of glass beads, phenol, chloroform, and SDS, as described (3), The Si probe was made by PCR amplification of a fragment followed by two further cold phenol extractions and three containing the promoter and about 850 nt of the thrS gene phenol/chloroform extractions. It was finally precipitated in cloned in pBluescript, using the same labeled oligonucleotide ethanol and resuspended in an appropriate volume of diethyl- as for the RT reactions and the Universal primer as the pyrocarbonate (DEPC)-treated water. upstream oligonucleotide (see Materials and Methods). The Reverse Transcriptase (RT) Assays. RT assays were carried band corresponding to the RT, stop between the T-box and the out as described (10) by using 15 ,ug of total RNA and about terminator was also a product of the Si nuclease reaction (Fig. 1 pmol of 5'-end labeled oligonucleotide. RNA and oligonu- 1B, lane 2), confirming that this 5' end exists in vivo. Control cleotide were heated together at 65°C for 5 min, and then reactions with total E. coli tRNA or the probe alone (lanes 3 frozen in a mixture of dry ice and ethanol and allowed to thaw and 4) did not show a band in this position. Since no such 5' on ice. Reactions contained 2 units of avian myeloblastosis end is observed in in vitro transcription experiments, and the virus RT (Eurogentec, Brussels) and were allowed to run for 160 bp upstream of this site show no promoter activity in vivo 30 min at 48°C. Oligonucleotides were synthesized by Oligo- when fused to lacZ (data not shown), the possibility of Express (Montreuil, France), and the sequences are available transcription initiation from a second gene promoter is elim- on request. inated. We thus concluded that the shorter thrS transcript is a S1 Nuclease Assays. S1 probes were amplified by PCR from cleavage product of the full-length mRNA. plasmids containing either part of the thrS gene or thrS-lacZ The Processed thrS Messenger RNA Is More Stable Than fusions. Probes complementary to the thrS leader and part of the Full-Length Transcript, Particularly Under Starvation the structural gene, which are specific for the native thrS gene Conditions. The processed transcript is likely to have a termi- transcripts, were amplified from pHMS2, a pBluescript- nator structure at both its 5' and its 3' end. Since stable derived plasmid described previously (see figure 3 in ref. 11). secondary structures at the ends of messenger RNAs have Thirty picomol of a 5'-end labeled oligonucleotide (28-mer) been associated with increased stability in E. coli (12-15), we starting at position +503 of the thrS transcript and 30 pmol of examined the stability of the thrS full-length and processed the Universal primer were used in the PCR. Probes specific for mRNAs under normal and starvation conditions. RNA was the wild-type and mutant thrS-lacZ fusions were amplified isolated from B. subtilis strains at 2-min intervals following the from their respective plasmids pHMS69 (wild type; this paper), addition of rifampicin, which prevents further transcription pHMS34 (stop codon mutant), and pHMS29 (deletion of initiation, and subjected to S1 nuclease analysis as above (Fig.
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